Molecular phenotyping and functional assessment of smooth muscle like-cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AA) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMC), genes encoding proteins of the TGFβ signaling pathway, and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contractility (ACTA2, MYH11), TGFβ signaling (SMAD3) and a dominant negative (DN) and two haploinsuficient variants (HI) in the extracellular matrix elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFβ signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be a promising tool to help reclassification of variants of unknown clinical significance in AA related genes.